Single nucleotide polymorphisms (SNPs) are the most common type of variation in the human genome. Mutations are also usually SNPs but the term is normally reserved for those with a frequency rarer than 1% or where there is a known functional, disease-causing role for the variation (Gibson N J, 2006, Clin Chim Acta. 363(1-2):32-47). There are many applications for genotyping polymorphisms and detecting rare mutations. The detection of rare variants is important for the early detection of pathological mutations, particularly in cancer. For instance, detection of cancer-associated point mutations in clinical samples can improve the identification of minimal residual disease during chemotherapy and detect the appearance of tumour cells in relapsing patients The measurement of mutation load is also important for the assessment of environmental exposure to mutagens, to monitor endogenous DNA repair, and to study the accumulation of somatic mutations in aging individuals. Additionally, more sensitive and quantitative methods to detect rare variants can revolutionise prenatal diagnosis, enabling the characterisation of foetal cells present in maternal blood. A vast number of methods have been introduced, but no single method has been widely accepted. Many methods for detecting low-frequency variants in genomic DNA use the polymerase chain reaction (PCR) to amplify mutant and wild-type targets. The PCR products are then analysed in a variety of ways, including sequencing, oligonucleotide ligation, restriction digestion, mass spectrometry or hybridization with allele-specific oligonucleotides to identify the variant against the background of wild-type DNA. Other methods use allele-specific PCR to selectively from the low-frequency variant, with or without additional selection. For example, by digesting PCR products with a restriction enzyme that specifically cleaves the wild-type product. Current approaches have inherent limitations due to the lack of total specificity of allele-specific primers during PCR, which creates false positives. As a result, all current approaches have limited sensitivity and accuracy (review in Jeffreys A J and May C A, 2003 Genome Res. 13(10):2316-24).
The real-time polymerase chain reaction (PCR) can be used for SNP genotyping. It is carried out in a closed-tube format and it is quantitative. Several methods are currently available for performing real-time PCR, such as utilising TaqMan probes (U.S. Pat. Nos. 5,210,015 and 5,487,972, and Lee et al., Nucleic Acids Res. 21:3761-6, 1993), molecular beacons (U.S. Pat. Nos. 5,925,517 and 6,103,476, and Tyagi and Kramer, Nat. Biotechnol. 14:303-8, 1996), self-probing amplicons (scorpions) (U.S. Pat. No. 6,326,145, and Whitcombe et al., Nat. Biotechnol. 17:804-7, 1999), Amplisensor (Chen et al., Appl. Environ. Microbiol. 64:4210-6, 1998), Amplifluor (U.S. Pat. No. 6,117,635, and Nazarenko et al., Nucleic Acids Res. 25:2516-21, 1997, displacement hybridization probes (Li et al., Nucleic Acids Res. 30:E5, 2002); DzyNA-PCR (Todd et al., Clin. Chem. 46:625-30, 2000), fluorescent restriction enzyme detection (Cairns et al. Biochem. Biophys. Res. Commun. 318:684-90, 2004) and adjacent hybridization probes (U.S. Pat. No. 6,174,670 and Wittwer et al., Biotechniques 22:130-1, 134-8, 1997). These methods are generally not suitable for mutation detection due to low accuracy, however.
The unifying problem behind all of these PCR approaches for detecting rare variants is replication infidelity during amplification or impreciseness of probe hybridisation. This is apparent in a popular mutation detection method described by Newton et al (Nucleic Acids Res. 17:2503-16, 1989; U.S. Pat. No. 5,595,890). This system, an amplification refractory mutation system (ARMS), exploits allele-specific primers that are used for a PCR reaction. Mispriming during amplification often yields inaccurate results.
EP0663447 relates to a method of detecting a polynucleotide by hybridizing a polynucleotide of known nucleotide sequence with a nuclease-resistant oligonucleotide primer having a sequence complementary to a part of said polynucleotide, then adding at least one kind of deoxynucleoside triphosphate, DNA polymerase and nuclease thereto, synthesizing a complementary strand being a nucleotide species located adjacent to the 3′-terminal of said primer and complementary to said polynucleotide, followed by decomposition thereof, the synthesis and decomposition of said complementary strand being repeated one or more times, and detecting the resulting pyrophosphoric acid or deoxynucleoside monophosphate
Nevertheless, it will be appreciated that the provision of nucleic acid detection methods that are both accurate and sensitive would provide a contribution to the art.